Objective: Traumatic brain injury (TBI) patients often experience massive bleeding and require blood transfusion. However, the storage duration of the transfused blood may affect the prognosis of these patients. This study explored the influence of exosomes derived from fresh and aged blood on the prognosis of rats with TBI, so as to provide theoretical support for the blood transfusion management of TBI patients. Methods: Exosomes were isolated from red blood cell (RBC) suspensions stored for 1 week and 5 weeks using ultracentrifugation method. The size, morphology and surface markers of the exosomes were identified by nanoparticle flow cytometry, transmission electron microscopy and Western blotting, respectively. A rat model of TBI was constructed using a mechanical impactor for brain injury. After the successful establishment of the model, exosomes from RBC suspensions stored for 1 week and 5 weeks were injected into the extracellular space of rat brain cells using a stereotactic syringe. Cerebral edema at day 1, 3, 7 and 14 were recorded through cranial magnetic resonance imaging (MRI) scans. Magnetic tracing technology (the tracer was Gd-DTPA solution) was used to evaluate the drug metabolism level in the extracellular space of brain cells of TBI rats. The cranial magnetic resonance imaging was scanned every 15 or 30 minutes, and the recording lasted for a total of 240 minutes. The magnetic images were imported into the 3D-Slicer software in Dicom data format for analysis. Mass spectrometry technology was used to analyze the differential proteins of exosomes from RBC suspensions stored for 1 week and 5 weeks, and functional prediction was carried out to explore the possible mechanisms by which exosomes affect the prognosis of TBI. Results: After injection of exosomes into TBI rats, the areas of cerebral edema on the day 1, 3, 7, and 14 were all significantly higher in the rats treated with exosomes from 5-week-stored RBC suspensions, with peak cerebral edema occurring at day 3. The diffusion volume of the tracer was significantly higher in TBI rats than in normal rats, which implied there was a disorder in the structure of the traumatic brain tissue in TBI rats. Compared with the rats injected with exosomes from 1-week-stored RBC suspensions, those treated with exosomes from 5-week-stored RBC suspensions showed increased tracer diffusion volume within 120 minutes. Mass spectrometry analysis identified 81 differentially expressed proteins between exosomes from RBC suspensions stored for 5 weeks vs 1 week. Among them, 93.83% (76/81) proteins had increased expression levels. The neurodegeneration-related pathways were among the most enriched pathways for upregulated proteins. Conclusion: The exosomes from aged RBC suspensions can lead to exacerbated cerebral edema, disrupted extracellular space, and suppressed metabolic rate in TBI rats, suggesting that transfusion of aged RBC suspensions may have adverse effects on TBI patients.

Diabetic cardiomyopathy is a distinct form of cardiomyopathy that can lead to heart failure, arrhythmias, cardiogenic shock, and sudden death. It has become a major cause of mortality in diabetic patients. The pathogenesis of diabetic cardiomyopathy is complex, involving increased oxidative stress, activation of inflammatory responses, disturbances in glucose and lipid metabolism, accumulation of advanced glycation end products (AGEs), abnormal autophagy and apoptosis, insulin resistance, and impaired intracellular Ca²⁺ homeostasis. Recent studies have shown that adenosine monophosphate-activated protein kinase (AMPK) plays a crucial protective role by lowering blood glucose levels, promoting lipolysis, inhibiting lipid synthesis, and exerting antioxidant, anti-inflammatory, anti-apoptotic, and anti-ferroptotic effects. It also enhances autophagy, thereby alleviating myocardial injury under hyperglycemic conditions. Consequently, AMPK is considered a key protective factor in diabetic cardiomyopathy. As part of diabetes prevention and treatment strategies, both pharmacological and exercise interventions have been shown to mitigate diabetic cardiomyopathy by modulating the AMPK signaling pathway. However, the precise regulatory mechanisms, optimal intervention strategies, and clinical translation require further investigation. This review summarizes the role of AMPK in the prevention and treatment of diabetic cardiomyopathy through drug and/or exercise interventions, aiming to provide a reference for the development and application of AMPK-targeted therapies. First, several classical AMPK activators (e.g., AICAR, A-769662, O-304, and metformin) have been shown to enhance autophagy and glucose uptake while inhibiting oxidative stress and inflammatory responses by increasing the phosphorylation of AMPK and its downstream target, mammalian target of rapamycin (mTOR), and/or by upregulating the gene expression of glucose transporters GLUT1 and GLUT4. Second, many antidiabetic agents (e.g., teneligliptin, liraglutide, exenatide, semaglutide, canagliflozin, dapagliflozin, and empagliflozin) can promote autophagy, reverse excessive apoptosis and autophagy, and alleviate oxidative stress and inflammation by enhancing AMPK phosphorylation and its downstream targets, such as mTOR, or by increasing the expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor‑α (PPAR‑α). Third, certain anti-anginal (e.g., trimetazidine, nicorandil), anti-asthmatic (e.g., farrerol), antibacterial (e.g., sodium houttuyfonate), and antibiotic (e.g., minocycline) agents have been shown to promote autophagy/mitophagy, mitochondrial biogenesis, and inhibit oxidative stress and lipid accumulation via AMPK phosphorylation and its downstream targets such as protein kinase B (PKB/AKT) and/or PPAR‑α. Fourth, natural compounds (e.g., dihydromyricetin, quercetin, resveratrol, berberine, platycodin D, asiaticoside, cinnamaldehyde, and icariin) can upregulate AMPK phosphorylation and downstream targets such as AKT, mTOR, and/or the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), thereby exerting anti-inflammatory, anti-apoptotic, anti-pyroptotic, antioxidant, and pro-autophagic effects. Fifth, moderate exercise (e.g., continuous or intermittent aerobic exercise, aerobic combined with resistance training, or high-intensity interval training) can activate AMPK and its downstream targets (e.g., acetyl-CoA carboxylase (ACC), GLUT4, PPARγ coactivator-1α (PGC-1α), PPAR-α, and forkhead box protein O3 (FOXO3)) to promote fatty acid oxidation and glucose uptake, and to inhibit oxidative stress and excessive mitochondrial fission. Finally, the combination of liraglutide and aerobic interval training has been shown to activate the AMPK/FOXO1 pathway, thereby reducing excessive myocardial fatty acid uptake and oxidation. This combination therapy offers superior improvement in cardiac dysfunction, myocardial hypertrophy, and fibrosis in diabetic conditions compared to liraglutide or exercise alone.

BACKGROUND:Studies demonstrated that miR-135a-5p was highly expressed in mouse embryonic palatal mesenchymal cells with cleft palate induced by dexamethasone.The primary cilium and its mediated Shh signaling pathway were involved in the autophagy of mouse embryonic palatal mesenchymal cells.It is speculated that miR-135a-5p may regulate autophagy in mouse embryonic palatal mesenchymal cells through primary cilia and its mediated Shh signaling pathway. OBJECTIVE:To investigate the regulatory effect of miR-135a-5p on autophagy of mouse embryonic palatal mesenchymal cells. METHODS:In vitro,palatal mesenchymal cells from C57BL/6J mouse embryos were extracted and cultured.Cell transfections were set up as follows:(1)the cells were divided into control group,miR-135a-5p negative control group and miR-135a-5p mimic group;(2)NC+miR-NC group,KIF3B overexpression group,and miR-135a-5p+KIF3B group:qRT-PCR was performed to verify transfection efficiency of miR-135a-5p and KIF3B.A transmission electron microscope was used to observe the number of autophagosome/autophagolysosome in the cells of each group.The degree of fluorescence expression of autophagy marker LC3B was determined by the immunofluorescence technique.The protein expression of KIF3B,LC3 and P62 was determined by western blot assay.(3)The cells were divided into miR-135a-5p negative control group,and SAG treated group,and SAG+miR-135a-5p group.qRT-PCR was used to detect the mRNA expression levels of Gli3,a key transcription factor downstream of Shh signaling.The protein expressions of autophagy-related proteins LC3 and P62 were detected by western blot assay. RESULTS AND CONCLUSION:(1)After overexpression of miR-135a-5p,the number of autophagosome/autophagolysosome was significantly increased(P<0.01).The fluorescence density of LC3B increased significantly(P<0.01);the protein expression of KIF3B and P62 decreased(P<0.01),and the protein expression of LC3 increased.(2)After overexpression of KIF3B,the number of autophagosome/autophagolysosome was significantly decreased(P<0.01);the fluorescence density of LC3B was decreased(P<0.01);the protein expression of P62 was increased(P<0.01),and the protein expression of LC3 was decreased(P<0.01).Targeted expression of KIF3B was inhibited by miR-135a-5p(P<0.01);the number of autophagosome/autophagolysosome,the fluorescence intensity of LC3B as well as the protein expression of LC3 were reversed(P<0.01)and the protein expression of P62 was decreased(P<0.01).(3)SAG significantly increased the mRNA expression of Gli3(P<0.01),increased the protein expression of P62(P<0.01),and decreased the protein expression of LC3(P<0.01).When miR-135a-5p was added,Gli3 mRNA expression was significantly decreased(P<0.01);P62 protein expression was decreased(P<0.01),and LC3 protein expression was reversed(P<0.01).(4)These results indicate that miR-135a-5p targets the inhibition of KIF3B and promotes autophagy in mouse embryonic mesenchymal cells possibly by negatively regulating the Shh signaling pathway.

Objective Reliability and validity of the Chinese version of disability attitude scales(DAS-CN)toward disabled persons were created and tested to provide an assessment instrument for measuring the attitude of medical staff toward disabled persons in China.Methods Authorised by the author of DAS in August 2020,based on BRISLIN translation model,the English version of DAS was translated into Chinese followed by back translation,cultural debugging and then put it into pre-experiment in September 2020.The reliability and validity of the finalised DAS-CN were further tested in a survey with 400 randomly selected medical staff in rehabilitation from 8 general hospitals in Jinzhou,Panjin,Yingkou and Fushun in Liaoning Province,China by using the convenience sampling method in March 2021.Results A total of 357 surveyees completed the survey.The localised DSA-CN was composed of 4 dimensions with a total of 20 items,including 4 items in clinical knowledge and skills,4 in clinical responsibility,8 in clinical behaviour and 4 in emotional response.The Cronbach α coefficient of the scales was 0.943,with the split-half reliability and test-retest reliability at 0.824 and 0.899,respectively.The Cronbach α coefficient of each dimension was 0.843～0.944,and the split half reliability was 0.854～0.904.The test-retest reliability ranged from 0.701 to 0.913.The KMO value of exploratory factor analysis was 0.921.The Bartrett spherical test value was 5534.981(P<0.01).The total explanatory rate of variation was 73.050%.Conclusion The Chinese version of Disability Attitude Scales(DSA-CN)has good reliability and validity.Therefore,DSA-CN can be used as an instrument in investigation of the current status about the attitudes towards the disabled persons among the medical staff in China.

Sesquiterpenoids are widely found in nature, while nitrobenzoyl sesquiterpenoids are relatively rare. Twelve natural nitrobenzoyl sesquiterpenoids were all derived from marine Aspergillus fungi, which are typical natural products with marine characteristics. These natural products exhibit good antitumor, antiviral, and inhibition of osteoclast differentiation activity, especially in the treatment of osteoclast-related diseases, showing good medicinal development value. This article reviews the natural product sources, chemical structure, chemical synthesis, biosynthesis, bioactivity, and pharmacological mechanisms of nitrobenzoyl sesquiterpenoids and predicts and discusses their absorption, distribution, metabolism, excretion, toxicity (ADME/T), and drug-likeness, providing a comprehensive understanding of the natural products of nitrobenzoyl sesquiterpenoids from marine sources and their potential for pharmaceutical development.

Objective To investigate the role of programmed necrosis pathway in testicular tissue damage in rats with erectile dysfunction(ED)and the mechanism of sildenafil intervention.Methods An ED rat model was established by high-fat chow feeding,and the rats were randomly divided into model group,experimental group,interleukin 18 group,and experimental+interleukin 18 group;10 rats were randomly selected as the normal control group.The experimental group was gavaged with 20 mg·kg-1sildenafil;the interleukin 18 group was injected intraperitoneally with 0.2 μg·kg-1interleukin 18 recombinant protein;the experimental+interleukin 18 group was gavaged with an equal amount of sildenafil,and the experimental+interleukin 18 group was injected intraperitoneally with an equal amount of interleukin 18 recombinant protein;the normal control and model groups were given with an equal amount of 0.9％NaCl by gavage and intraperitoneally injected with an equal amount of saline;and the experimental group was injected with an equal amount of 0.9％NaCl.Interleukin 18 group was gavaged with an equal amount of 0.9％NaCl,and the five groups of rats were administered once a day at regular intervals for 14 consecutive days.Reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression of receptor-interacting protein kinase 1(RIP1),receptor-interacting protein kinase 3(RIP3),mixed-spectrum kinase structural domain-like protein(MLKL),and calcium-calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)in rat testis tissues.Results The relative expression levels of RIP1 protein in the normal control group,model group,experimental group,interleukin 18 group and experimental+interleukin 18 group were 0.58±0.05,0.99±0.09,0.71±0.05,1.23±0.07 and 0.81±0.07;the relative expression levels of RIP3 protein were 0.61±0.05,1.05±0.10,0.77±0.04,1.19±0.07 and 0.84±0.08,respectively;the relative expression levels of MLKL protein were 0.63±0.05,1.13±0.08,0.79±0.05,1.30±0.02 and 1.00±0.04,respectively;the relative expression levels of CaMK Ⅱ protein were 0.54±0.04,1.12±0.07,0.77±0.05,1.36±0.04 and 1.00±0.07;the differences of the above indexes were statistically significant when the model group was compared with the normal control group,the experimental group was compared with the model group,the interleukin 18 group was compared with the model group,and the experimental+interleukin 18 group was compared with the interleukin 18 group(all P＜0.05).Conclusion The RIP1/RIP3-mediated necroptosis pathway plays multiple regulatory roles in testicular injury in ED rats,and sildenafil may improve testicular function in rats by inhibiting the above necroptosis signaling pathway.

Objective To investigate the expression of miR-31a-5p in myocardial infarction (MI) mice and its potential mechanism. Methods A dataset was downloaded from the gene expression database, and miR-31a-5p and its predicted target gene hypoxia-inducible factor-1α (HIF-1α) were screened using bioinformatics methods. The MI model was established by ligating the left anterior descending branch of the coronary artery in C57BL/6J male mice which were randomly divided into sham and MI groups (n=6 in each group). The in vitro hypoxic cell model was induced by treatment of H9c2 cells with cobalt chloride (CoCl2) and divided into a control group, a model group, a NC group, a miR-31a-5p mimic group and a miR-31a-5p inhibitor group. The degree of myocardial tissue fibrosis was stained by Masson and analyzed. The expression levels of miR-31a-5p and HIF-1α mRNA in mouse myocardial tissues and H9c2 cells were detected by qRT-PCR. Western blotting was used to detect the expression levels of B-cell lymphoma 2 (Bcl-2), cleaved-caspase 3 apoptotic protein in mouse myocardial tissues and HIF-1α and apoptotic protein in H9c2 cells, respectively. The dual luciferase reporter gene assay was used to verify the targeting relationship between miR-31a-5p and HIF-1α. Results Masson staining showed significantly increased fibrosis in MI mice (P<0.000 1); miR-31a-5p, cleaved-caspase 3 were significantly elevated and Bcl-2 was decreased in MI mice and CoCl2 treated H9c2 (P<0.05). The results of dual luciferase reporter assay showed that the relative luciferase activity of miR-31a-5p mimic cotransfected with HIF-1α-3'-UTR WT plasmid was reduced (P<0.000 1); miR-31a-5p mimic decreased HIF-1α expression and increased apoptotic protein levels in CoCl2 induced H9c2 cells (both P<0.05), while miR-31a-5p exerted the opposite effect. Conclusion miR-31a-5p can aggravate apoptosis in myocardial ischemia by targeting HIF-1α.

Objective This study aims at establishing a teaching catalog and content for breast rare dis-eases and developing the syllabus for the breast rare disease using integrated multimodality of case-/problem-/resource-based learning(CBL+PBL+RBL).Methods By conducting bibliometrics co-occurrence analysis,we collected 6291 articles on breast rare disease published from January,1975 to June,2024.Additionally,we re-trieved the Textbook on Rare Diseases,the Catalog of Chinese Rare Disease,and Second Batch of Rare Dis-ease Catalog and then decided the teaching content.Results From 16,387 keywords,1000(6.1％)keywords were identified through co-occurrence analysis,including 50(0.3％)candidate diseases.These were classified into three categories:rare primary breast diseases,rare genetic mutation-related diseases associated with breast cancer,and rare systemic multi-system diseases involving the breast.From the candidate list,20(0.1％)rare primary breast diseases were further selected for their notable clinical teaching significance,and significant multi-systemic diseases affecting the breast,whether related to gene mutations or not.Teaching plans were draf-ted using a diversified parallel teaching approaches,taking into account the characteristics of different diseases and the focus of different teaching methods.Conclusions This study initiated the development of the teaching content for breast rare diseases and developed the teaching syllabus using the CBL+PBL+RBL integrated multi teaching model and targeting each rare breast disease for the critical point for teaching.

Objective:To explore the role of metagenomic next-generation sequencing (mNGS) in the diagnosis of pathogens in primary infectious diseases of the spine (IDS) and to reveal its pathogen spectrum.Methods:This is a retrospective multi-center case series study. Clinical data of 380 patients with primary IDS who were treated at four medical centers in China from December 2019 to April 2024 were retrospectively analyzed. Among them, 82 cases were from the Department of Spine Surgery at the Affiliated Hospital of Qingdao University, 129 cases were from the Orthopedics Section Ⅱ (Bone Infection), Public Health Clinical Center Affiliated to Shandong University, 112 cases were from the Department of Spine Surgery, Fuzhou Second General Hospital, and 57 cases were from the Department of Orthopedic Surgery, Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. There were 238 males and 242 females, with an age of (61.4±13.1) years (range: 10 to 91 years). Specimens from the site of spinal infection were obtained for pathogen culture, pathological examination, and mNGS detection preoperatively or intraoperatively in all patients. The number, types, and positive rates of pathogens detected by the two methods were analyzed and compared using the Chi-square test.Results:Among the 380 patients, 320 had confirmed pathogenic bacteria, with the highest proportion being pyogenic bacterial infections, accounting for 76.9% (246/320). The most common pathogen was Staphylococcus aureus, accounting for 22.8% (73/320). Brucella accounted for 13.8% (44/320); Mycobacterium tuberculosis accounted for 6.3% (20/320). Fungal infections accounted for 3.4% (11/320), mainly Aspergillus and Candida. In addition, Mycoplasma was detected in 3 cases (0.9%) and Benacox body in 4 cases (1.2%). The pathogen spectrum constructed by mNGS covered 46 types of pathogens, higher than the 22 types detected by traditional methods. The positive rate of mNGS was 80.8% (308/381), significantly higher than the 27.9% (106/381) of traditional methods ( χ2=182.53, P<0.01). Conclusions:mNGS improves the positive rate of pathogen diagnosis in IDS, detecting a broader spectrum of pathogens, and serves as a valuable complement to traditional diagnostic methods. Combining both methods in the diagnosis of IDS can maximize detection rates, providing robust evidence for precise anti-infective treatment.

To characterize human antibodies against low-calcium response V(LcrV)antigen of Yersinia pestis,the mono-clonal antibodies were screened and assayed.Antibody gene was derived from peripheral blood mononuclear cells of the vaccin-ees immunized by plague subunit vaccine in phase Ⅱb clinical trial.Human ScFv antibody library was constructed by phage dis-play.After panning library by using recombinant LcrV antigen,antibody variable genes were sequenced and converted into IgG1 format to evaluate its binding specificity and relevant parameters.An anti-plague human ScFv antibody library was estab-lished contained 7.54× 108 independent clones.After panning by LcrV antigen,3 human antibodies named as RV-B4,RV-D1 and RV-E8,respectively,were identified.Using indirect enzyme-linked immunosorbent assay(ELISA)and Western blot(WB),the specific bindings of the mAbs to LcrV antigen were confirmed.The dissociation constant(KD)of them to LcrV is 2.1 nmol/L,1.24 nmol/L and 42 nmol/L,respectively.Minor protective efficacy was found among 3 human antibodies in Y.pestis 141-infected mice.Three anti-LcrV monoclonal antibodies generated from immunized vaccinees were binding specific antibod-ies and could not block plague infection in mice.These antibodies are the potential candidate reagents for basic research of plague immunity and the application of plague diagnosis.